GABA B Receptor Agonist Baclofen Has Non-Specific Antinociceptive Effect in the Model of Peripheral Neuropathy in the Rat

Physiol. Res. 3: 31-3, GABA B Receptor Agonist Baclofen Has Non-Specific Antinociceptive Effect in the Model of Peripheral Neuropathy in the Rat M. FRANĚK, Š. VACULÍN, R. ROKYTA Department of Normal, Pathological and Clinical Physiology, Third Faculty of Medicine, Charles University, Prague, Czech Republic Received July, 3 Accepted November 7, 3 Summary Baclofen, which is a specific agonist of the metabotropic GABA B receptor, is used in clinical practice for the treatment of spasticity of skeletal muscles. It also exerts an analgesic effect, but this effect is still not clear and especially controversial in neuropathic pain. In this work, we studied the antinociceptive effects of baclofen in a model of chronic peripheral neuropathic pain loose ligation of the sciatic nerve (chronic constriction injury, CCI). As controls we used sham-operated animals. The changes of thermal pain threshold were measured using the plantar test 1- days after the operation. The obtained results suggest that baclofen increases pain threshold in both groups. The antinociceptive effect of baclofen was dose-dependent and the maximum response without motor deficits was observed at a dose of 1 mg/kg s.c. In the rats with CCI, significant differences between affected (ipsilateral) and contralateral hind paw were present. This difference was dose-dependent, the highest value (.±1.37 s) was found at the dose of mg/kg. Based on our results and previous findings it could be summarized that baclofen has antinociceptive action, which is attenuated in the model of chronic neuropathic pain probably due to the degeneration of GABA interneurons after chronic constriction injury. Key words Baclofen GABA B receptor Chronic constriction injury Antinociception Rat Introduction It is generally accepted that γ-aminobutyric acid (GABA) is involved in spinal antinociception (Sawynok 1987). GABAergic neurons found in laminae I-III are mainly inhibitory interneurons (Gobel 1978). In the superficial layers of the dorsal horn there are also present both ionotropic GABA A and metabotropic GABA B receptors (Price et al. 198, Bowery et al. 1987). The activation of GABA B receptors leads to the inhibition of presynaptic mediator release (blockade of voltage-gated calcium channels) (Wojcik et al. 199) and to the inhibition of postsynaptic neuronal activity (activation of G-protein-gated inwardly rectifying potassium channels and inhibition of adenylyl cyclase activity) (Otis et al. 1993, Knight and Bowery 199). Baclofen (-amino-3-[cholophenyl] butyric acid; Lioresal) is a specific agonist of heptahelical GABA B receptor. Baclofen is used for treating the spasticity but besides this action it also induces analgesia in a number PHYSIOLOGICAL RESEARCH ISSN 8-88 Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic Fax + 1 1 E-mail: physres@biomed.cas.cz http://www.biomed.cas.cz/physiolres

3 Franěk et al. Vol. 3 of animal pain models. The main antinociceptive mechanism of baclofen is thought to be the glutamate release inhibition from Aδ and C primary afferent terminals in substantia gelatinosa (Ataka et al. ). Moreover, baclofen decreases the NK-1 receptor expression in the spinal dorsal horn (Enna et al. 1998). Our study was carried out in order to determine the effect of baclofen on pain threshold for thermal stimulation in the model of chronic peripheral neuropathic pain loose ligation of sciatic nerve (chronic constriction injury, CCI) in the rat (Bennett and Xie 1988). Recent studies have shown that baclofen has controversial antinociceptive effects in the models of persistent neuropathic pain (Smith et al. 199, Hwang and Yaksh 1997, Hao et al. 1999, Patel et al. 1). In our experiment, the antinociceptive effect of baclofen was compared with morphine at the dose of mg/kg s.c. This dose is recommended for the sedation in rat (Ben et al. 199) and we used it as a reference drug. Methods Animals Adult male Wistar rats (Velaz, Prague, Czech Republic) weighing 1- g were housed with free access to food and water and maintained under a regime with 1 h of light and 1 h of darkness per day. The mean temperature was ± C and the relative humidity equaled to ±1 %. The acclimation period was days long. This experiment was approved by the Committee for Animal Care and Use of the Third Faculty of Medicine, Charles University, Prague, and conducted according to the guidelines of the Ethics Committee of the International Association for the Study of Pain (Zimmermann 1983). Surgical procedures Persistent neuropathic pain in rats was evoked by chronic constriction of sciatic nerve according to the model of Bennett and Xie (1988). Briefly, under pentobarbital anesthesia ( mg/kg, supplemented when necessary), the right sciatic nerve was exposed and lightly ligated with four ligatures of chromic catgut. In shamoperated animals, the right sciatic nerve was exposed in the same manner, but not ligated. In all animals the left sciatic nerve remained unoperated. Pain testing Antinociceptive effects of drugs were determined according to the latency (in seconds) of limb withdrawal to the noxious thermal stimulation (Plantar test, Ugo Basil, Comerio, Italy). Cut-off value of plantar test was set to s to prevent limb injury. The pain thresholds were tested 1- days following the surgical procedure. Drug application All the drugs were administrated by subcutaneous injection in the following doses: baclofen (Sigma) 1,, 1, 1, and 3 mg/kg, morphine (Léčiva Prague, Czech Republic) mg/kg and saline in the same volume as baclofen. Baclofen was administrated in suspension (1 % gum accacia (Sigma) in.9 % saline) in volume of ml/kg. Pain thresholds were evaluated three to five times for each hind limb 1,, 3,, and 9 min after drug administration. Data analysis Data are expressed as mean ± S.E.M. of - animals in each group. To determine the statistical significance, the ANOVA test (Statistica., StatSoft Inc.) was used. Differences between means were considered statistically significant if p<.. Results Baclofen increases thermal pain threshold in control animals. When administered subcutaneously (s.c.) baclofen showed significant pain threshold elevation in sham operated animals. This effect was observed in the dose of mg/kg. In the dose of 1 mg/kg s.c. the obtained plantar test latency was not significantly different when compared to saline (Fig. 1A). Morphine in the dose of mg/kg s.c. was several times more potent in this group the cut-off value s was overshot. There were no significant differences between plantar test latency of ipsilateral (sham-operated) and contralateral (unaffected) hind limb in any of the tested compounds. Baclofen also elevated the plantar test latencies in the model of peripheral neuropathy. The animals with chronic constriction of the sciatic nerve were sensitive to baclofen in a similar manner as the sham-operated ones (Fig. 1B). Of course, the difference compared to shamoperated animals was in the pain thresholds between ipsilateral (ligated) and contralateral (unaffected) hind limbs. The pain threshold was lower in the case of ipsilateral limb. This was observed after the administration of both saline and baclofen. After administration of 1 mg/kg baclofen the plantar test latency was not significantly different compared to saline, but this difference was found in animals with the dose of

Antinociceptive Effect of Baclofen 33 mg/kg. Morphine in the dose of mg/kg overshot the cut-off value of s. latency [s] latency [s] 1 1 8 1 1 1 1 8 A B saline baclofen 1 mg/kg contralat. Fig. 1. Plantar test latencies (mean ± S.E.M.; n = -) after drug administration in control (A) and ligated (B) animals. Baclofen in the dose of mg/kg s.c. increases the thermal pain threshold in both control and neuropathic animals. Hatched bars, left hind limbs latencies (contralateral to the sham-operated (A) or ligated (B) sciatic nerve); open bars, right hind limbs latencies (ipsilateral to the sham-operated (A) or ligated (B) sciatic nerve). After morphine administration cut-off value s was overshot. p<.; p <. compared to saline, ANOVA test. The elevation of the pain threshold by baclofen is dose-dependent. The changes of plantar test latencies were evaluated after following doses of baclofen: 1,, 1, 1, and 3 mg/kg s.c. As illustrated in Figure, the increase of the pain threshold was dose-dependent. The significant difference compared to saline was observed for the doses of, 1, 1, and 3 mg/kg. After the administration of baclofen in the dose of 3 mg/kg the cut-off value s was overshot. The motor deficit was observed for the doses of and 3 mg/kg. The dose-dependent effect of baclofen was observed in both ipsilateral (ligated) and contralateral (unaffected) hind limbs. baclofen mg/kg ipsilat. morphine mg/kg [s] 1 1 contralat. ipsilat. 1 1 1 baclofen [mg/kg] Fig.. Antinociceptive activity of baclofen in the model of peripheral neuropathy is dose-dependent. In both ligated and unaffected hind limbs baclofen increases the thermal pain threshold. Solid squares, left hind limbs latencies (contralateral to the ligated sciatic nerve); open circles, right hind limbs latencies (ipsilateral to the ligated sciatic nerve). Data are expressed as mean ± S.E.M. (n = -). p<. compared to saline; p<. compared to saline, ANOVA test. There are differences in baclofen effects on ligated and unaffected hind limbs. As mentioned in the previous paragraph, baclofen increases thermal pain threshold in a dose-dependent manner in both ligated and unaffected hind limbs. Moreover, the intensity of the increase (i.e. differences in the latencies between the two hind limbs for different doses, antinociceptive potential) differs between the two limbs. Figure 3 (gaps between ligated and unaffected limbs) shows that baclofen increases pain threshold significantly more in contralateral (unaffected) hind limb. These gaps are dosedependent with the maximum obtained value.±1.37 s for the dose of mg/kg. 1 Fig. 3. Plantar test latencies gaps (mean ± S.E.M.; n = -) between contralateral (unaffected) and ipsilateral (ligated) hind limbs. Values represent the difference between unaffected and ligated hind limbs for different doses of baclofen. These gaps are dose-dependent. 1 1 3 1 7 [s] baclofen [mg/kg]

3 Franěk et al. Vol. 3 Discussion The chronic ligation of sciatic nerve is a widely used model of peripheral neuropathic pain. It is generally accepted that this model may mimic many important characteristics of neurogenic pain in patients after peripheral nerve injury (Cui et al. 1997, Zarrindast et al. ). The plantar test enables measurement of the effects of various conditions on the defensive paw withdrawal reflex, which is under spinal and supraspinal control (Franěk et al. ). The fact that baclofen modulates the pain threshold in non-affected animals, has already been described (Malcangio et al. 1991). Our results confirm the antinociceptive effects of baclofen in the intact animals, and this effect is mediated at least partially by spinal cord neurons (Gobel 1978). It has been suggested that one of possible mechanisms of baclofeninduced antinociception is the inhibition of excitatory neurotransmitters released from primary afferent fibers (Aran and Hammond 1991). The present study shows that a subcutaneous injection of baclofen, a typical GABA B receptor agonist (Bowery 1993), increases the pain threshold for the thermal stimulation in CCI rats. Similar results have been previously described for the mechanical stimulation (Smith et al. 199, Patel et al. 1). Our results confirm some antinociceptive action of baclofen in the neuropathic rats. However, the main finding of the present study is that the baclofen-induced increase in the latency was significantly higher in the intact hind limbs compared to the ligated ones in CCI rats. It means that antinociceptive effect of baclofen is attenuated under neuropathic conditions. Although Smith et al. (199) found neither changes in the number of GABA B binding sites nor changes in the affinity of [ 3 H] GABA to GABA B receptors in the spinal cord after chronic constriction of the sciatic nerve, it has not been corroborated by later studies. Our results are likely to be explained by the findings of Castro-Lopes. They described the decrease in the number of GABA B binding sites (affinity of [ 3 H] GABA to GABA B receptor) in lamina II of the spinal cord - weeks after the sciatic nerve injury (Castro- Lopes et al. 199). It is also known that the damage of primary afferent fibers (CCI) might lead to a degeneration of spinal neurons (Sugimoto et al. 199), and that the number of GABA-immunoreactive cells in laminae I-III of the spinal cord significantly falls after sciatic nerve transsection (Castro-Lopes et al. 1993). On the other hand, the antinociceptive effect of baclofen was found in our study to be dose-dependent even in both hind limbs after CCI. The curves of the dose-dependence had similar shape in both the intact and injured hind limb with a shift of the injured limb curve to lower values. The dose-dependent effect in the injured hind limb suggests an absence of a saturation of GABAergic neurones. It is concluded that besides the decrease in the number of GABAergic neurons and/or binding sites, other factors might play an important role in the attenuation of the antinociceptive effect of baclofen by chronic neuropathy. Acknowledgements This work was supported by the Research Project J13/98:111, by the Neuropsychiatric Research Center LNB1 and by the grant No. NF 7-3/3 from Internal Grant Agency of the Ministry of Health of the Czech Republic. References ARAN S, HAMMOND DL: Antagonism of baclofen-induced antinociception by intrathecal administration of phaclofen or -hydroxy-saclofen, but not δ-aminovaleric acid in the rat. J Pharmacol Exp Ther 7: 3-38, 1991. ATAKA T, KUMAMOTO E, SHIMOJI K, YOSHIMURA M: Baclofen inhibits more effectively C-afferent than Aδ-afferent glutamatergic transmission in substantia gelatinosa neurons of adult rat spinal cord slices. Pain 8: 73-8,. BEN M, DIXON RL, ADAMSON RH: Anesthesia in the rat. Fed Proc 8: 1-17, 199. BENNETT GJ, XIE YK: A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 33: 87-17, 1988. BOWERY NG: GABA B receptor pharmacology. Annu Rev Pharmacol Toxicol 33: 19-17, 1993.

Antinociceptive Effect of Baclofen 3 BOWERY NG, HUDSON AL, PRICE GW: GABA A and GABA B receptor site distribution in the rat central nervous system. Neuroscience : 3-383, 1987. CASTRO-LOPES JM, TAVARES I, COIMBRA A: GABA decreases in the spinal cord dorsal horn after peripheral neurectomy. Brain Res : 87-91, 1993. CASTRO-LOPES JM, MALCANGIO M, PAN BH, BOWERY NG: Complex changes of GABA A and GABA B receptor binding in the spinal cord dorsal horn following peripheral inflammation or neurectomy. Brain Res 79: 89-97, 199. CUI JG, O'CONNOR WT, UNGERSTEDT U, LINDEROTH B, MEYERSON BA: Spinal cord stimulation attenuates augmented dorsal horn release of excitatory amino acids in mononeuropathy via a GABAergic mechanism. Pain 73: 87-9, 1997. ENNA SJ, HARSTAD EB, MCCARSON KE: Regulation of neurokinin-1 receptor expression by GABA B receptor agonists. Life Sci : 1-13, 1998. FRANĚK M, VACULÍN Š, HESS L, ROKYTA R: Antinociceptive effects of medetomidin and its combination with ketamine in rat. Physiol Res 1: 1P,. GOBEL S: Golgi studies of the neurons in layer II of the dorsal horn of the medulla (trigeminal nucleus caudalis). J Comp Neurol 18: 39-13, 1978. HAO JX, XU IS, XU XJ, WIESENFELD-HALLIN Z: Effects of intrathecal morphine, clonidine and baclofen on allodynia after partial sciatic nerve injury in the rat. Acta Anaesthesiol Scand 3: 17-13, 1999. HWANG JH, YAKSH TL: The effect of spinal GABA receptor agonists on tactile allodynia in a surgically-induced neuropathic pain model in the rat. Pain 7: 1-, 1997. KNIGHT AR, BOWERY NG: The pharmacology of adenylyl cyclase modulation by GABA B receptors in rat brain slices. Neuropharmacology 3: 73-71, 199. MALCANGIO M, GHELARDINI C, GIOTTI A, MALMBERG-AIELLO P, BARTOLINI A: CGP 338, a new GABA B antagonist, prevents antinociception and muscle-relaxant effect induced by baclofen. Br J Pharmacol 13: 133-138, 1991. OTIS TS, DE KONINCK Y, MODY I: Characterization of synaptically elicited GABA B responses using patch-clamp recordings in rat hippocampal slices. J Physiol Lond 3: 391-7, 1993. PATEL S, NAEEM S, KESINGLAND A, FROESTL W, CAPOGNA M, URBAN L, FOX A: The effects of GABA B agonists and gabapentin on mechanical hyperalgesia in models of neuropathic and inflammatory pain in the rat. Pain 9: 17-, 1. PRICE GW, WILKIN GP, TURNBULL MJ, BOWERY NG: Are baclofen-sensitive GABA B receptors present on primary afferent terminals of the spinal cord? Nature 37: 71-7, 198. SAWYNOK J: GABAergic mechanisms of analgesia: an update. Pharmacol Biochem Behav : 3-7, 1987. SMITH GD, HARRISON SM, BIRCH PJ, ELLIOTT PJ, MALCANGIO M, BOWERY NG: Increased sensitivity to the antinociceptive activity of (+/-)-baclofen in an animal model of chronic neuropathic, but not chronic inflammatory hyperalgesia. Neuropharmacology 33: 113-118, 199. SUGIMOTO T, BENNETT GJ, KAJANDER KC: Transsynaptic degeneration in the superficial dorsal horn after sciatic nerve injury: effects of a chronic constriction injury, transection, and strychnine. Pain : -13, 199. WOJCIK WJ, TRAVAGLI RA, COSTA E, BERTOLINO M: Baclofen inhibits with high affinity an L-type-like voltage-dependent calcium channel in cerebellar granule cell cultures. Neuropharmacology 9: 99-97, 199. ZARRINDAST M, VALIZADEH S, SAHEBGHARANI M: GABA B receptor mechanism and imipramine-induced antinociception in ligated and non-ligated mice. Eur J Pharmacol 7: -7,. ZIMMERMANN M: Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1: 19-11, 1983. Reprint requests MUDr. Miloslav Franěk, Ph.D., Charles University, Third Faculty of Medicine, Dept. of Normal, Pathological and Clinical Physiology, Ke Karlovu, 1 Prague, Czech Republic. E-mail: franek@lf3.cuni.cz